As a carrier of drugs, a liposome has the characteristics such as enhancing therapeutic efficacy, reducing adverse effects, target delivering, and delayed release. Especially where a liposome is used as the carrier of anti-tumor drug, the drug can be targetedly delivered to tumor area and thus has reduced toxicity and enhanced efficacy.
There are many anti-tumor drugs in clinical application which can be categorized into 5 groups: cytotoxic agents, hormones, biological response modifier, monoclonal antibodies and other anti-tumor drugs. Among them, cytotoxic agents capture the biggest market share, and they can be categorized into 5 groups according to mechanism of action: (1) drugs acting on DNA chemical structure, such as alkylating agents and platinum compounds; (2) drugs modifying nucleic acid synthesis, such as methotrexate and fluorouracil; (3) drugs acting on nucleic acid transcription, such as doxorubicin and epidoxorubicin; (4) drugs acting on tubulin synthesis, such as taxanes and vinca alkaloids; drugs acting on topoisomerase, such as camptothecin; (5) other cytotoxic drugs. Among them, the drugs of groups (2) and (4) are of cell cycle-specific character, can only kill cells in specific period of malignant tumor cell proliferation cycle. Vinorelbine and topotecan are of the groups and are intensively investigated in the present invention.
It is necessary to control the drug release from liposome with the aim of reducing toxicity and enhancing efficacy, where anti-tumor drug with cell cycle-specific character is prepared into liposome. In case of too fast drug releases from liposome, the following results will be incurred: (1) part of drug is released from liposome before reaching tumor area and is cleared from blood too quickly to reach tumor area; (2) in view that tumor cells are in different growth periods at the same time, the drug reaching tumor area can not kill cells out of specific periods, which induces greatly reduced exposure of the drug to tumor cells and has a poor therapeutic efficacy but induces toxic response of normal tissues. So it is important to control the drug release from liposome especially for the drugs with cell cycle-specific character.
The release of liposomal drug is influenced by diversified factors including particle size, lipid membrane composition, inner water phase and methods of drug loading, inter alia. Methods of drug loading include active drug loading and passive drug loading. Passive drug loading is generally suitable for lipid-soluble drugs, while active drug loading is generally suitable for water-soluble drugs. Since vinorelbine and topotecan are both water-soluble weak alkalescent drugs, active drug loading is chosen to prepare their liposomes. Three methods of active drug loading are commonly used in the art: pH gradient method, ammonium sulfate gradient method and complexation gradient method.
(1) pH gradient method:
This method is invented by Canadian investigators in the 1980's. They discovered that pharmaceutical alkaloids such as doxorubicin could be actively transported and specifically aggregated into liposomes in the presence of pH gradient. The first thing in the process of preparation is to choose inner water phase buffer and outer phase buffer, which is critical since the buffers directly determines the stability of drug in storage and the release of drug in vivo. A blank liposome is formed by hydration with inner water phase buffer. The thus-obtained blank liposome is further processed to reduce the particle size within a desired range. Next, outer phase of the liposome may be replaced by using the technical means such as cross flow dialysis, column chromatography and pH modulation, so as to form pH gradient between outer and inner transmembrane phases. The drug loading may be accomplished at an appropriate temperature after the transmembrane gradient is formed.
Also the transmembrane pH gradient can be formed using an ionophore. During the preparation of the blank liposome, divalent ion salt, such as manganese sulfate, is encapsulated into the liposome, and then the outer phase of liposome is replaced by a buffer containing an ionophore, such as A23187 and EDTA. The ionophore can specifically transport divalent ion to outside of membrane and transport H+ to inside of liposome. Use of the above method can also form pH gradient between inside and outside of the membrane.
The mechanism of drug loading by pH gradient has been intensively investigated. Among 3 anthracycline liposome preparations available in the market, 2 preparations are prepared by active drug loading using pH gradient.
(2) ammonium sulfate gradient method
Ammonium sulfate gradient method is invented by Israeli investigators in early 1990's. The preparation process in this method is similar to that in traditional pH gradient method. First, blank liposome is prepared by using ammonium sulfate buffer. Then, ammonium sulfate in outer phase of the liposome is removed by cross flow dialysis inter alia to form ammonium sulfate gradient between the inside and the outside of lipid membrane. Then drug loading is accomplished under the condition of heating. It is confirmed in initial research that the drug loading by ammonium sulfate gradient may be related to pH difference between the inside and the outside of the phospholipid membrane caused by transmembrane diffusion of free ammonia. However, it is shown by strict theoretical deduction that the drug loading using ammonium sulfate gradient method may be a complicated process of double-directional diffusion, and the formation of pH gradient may be merely one of the factors.
The advantage of ammonium sulfate gradient method lies in that approximately neutral pH of the ammonium sulfate aqueous solution could not induce hydrolyzation of excess phospholipid molecules, because a relatively high temperature is required if saturated phospholipid is used to prepare the liposome. The lipid is apt to hydrolyze when traditional pH gradient method is used. Moreover, the in vivo drug release of the liposome prepared using ammonium gradient method may be different.
(3) complexation gradient method
In this method, transition metal ion salt, such as copper sulfate or nickel sulfate is used in inner water phase buffer to prepare blank liposome. Next, metal ion outside the liposome is removed by cross flow dialysis among others to form the metal ion gradient between the inside and the outside of lipid membrane. Then drug loading is accomplished under the condition of heating. The mechanism of drug loading is that the drug forms a stable complex with transition metal ion in the inner water phase of liposome and is thus restrained within liposome.
Sulfobutyl ether-β-cyclodextrin (SBE-β-CD) is an ionized derivative of β-cyclodextrin (β-CD) developed by Cydex of US in 1990's, which is the product of substitution reaction of β-CD with 1,4-butane sultone. The substitution may occur at hydroxyl group of position 2, 3, 6 in glucose unit of SBE-β-CD. SBE-β-CD is an excellent pharmaceutical excipient having the advantages such as good water-solubility, low nephrotoxicity and low haemolysis, and is licensed by FDA as an excipient for injection.
SBE-β-CD has been so far used for solubilization by inclusion of insoluble drug, and has been used widely in various dosage forms such as injection, oral formualtion, topical formulation inter alia. Chakraborty used SBE-β-CD to investigate liposomal preparation of amphotericin B, with the aim of using solubilization by inclusion of insoluble drug by SBE-β-CD (Therapeutic and hemolytic evaluation of in-situ liposomal preparation containing amphotericin-B complexed with different chemically modified β-cyclodextrins. J Pharm Pharmaceut Sci. 2003 Vol. 6, No. 2).
Wang Zhixuan & Deng Yingjie, et al. (Advances in liposome entrapped drug cyclodextrin complex delivery systems, Journal of Shenyang Pharmaceutical University, 2006 Vol. 23) review world-wide researches of liposome entrapped drug cyclodextrin complex, which is prepared by making insoluble drug into water-soluble cyclodextrin complex and entrapping the complex into inner water phase of liposome. It is difficult for insoluble drug to enter inner water phase of liposome, while complexation-inclusion by cyclodextrin increase water-solubility of the insoluble drug, and thus it is easy to entrap the drug into liposome. The main aim of making drug into liposome entrapped drug cyclodextrin complex is to increase the solubility of insoluble drug and thus the drug loading.
As the first-line drugs in anti-tumor therapy, liposomal preparations of vinorelbine and topotecan have been intensively investigated. Now the drug loading of liposomal vinorelbine and topotecan have been investigated by many research groups. However, some problems rise such as the following:
Inex company of Canada achieves the drug loading by using sphingomyelin and cholesterol at a molar ratio of 55:45 as lipid membrane, using magnesium sulfate solution as inner water phase to prepare blank liposome, then transporting magnesium ion out of the liposomal membrane via the ionophore A23187 and transporting H+ to inside of liposome, and thus generating pH gradient. The thus-obtained liposomal vinorelbine has an encapsulation rate of more than 90%, and is stable when stored at 2-8° C. for one year (Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity. Journal of Pharmaceutical Sciences, 2005 Vol. 94 No. 5.)
A Canadian research group leaded by Bally uses 2 methods and obtains topotecan liposomes having high encapsulation rate. In the first method, DSPC and cholesterol are used as lipid membrane, manganese sulfate solution as inner water phase to prepare blank liposome. Then pH gradient is formed using the ionophore A23187 and the drug loading is achieved. The mechanism of this method is similar to that used by Inex company. The second method uses DSPC and cholesterol as lipid membrane, copper sulfate solution as inner water phase to prepare blank liposome. However, the loading of topotecan is accomplished without adding A23187, because a stable complex is formed between copper ion and topotecan. The principle used herein is just the complexation gradient method as described above. The disadvantage of this method is that remaining metal ion in the formulation may cause toxic effect in blood (An evaluation of transmembrane ion gradient-mediated encapsulation of topotecan within liposomes. Journal of Controlled Release. 96 (2004); Copper-topotecan complexation mediates drug accumulation into liposomes. Journal of Controlled Release. 114 (2006))
US investigators use distearoyl phosphatidyl choline (DSPC), cholesterol and distearoyl phosphatidyl ethanolamine-methoxyl-polyethylene glycol conjugate (DSPE-mPEG) as lipid membrane, use triethylamine (TA) salt of sucrose octasulfate as inner water phase to prepare blank liposome. Then TA sucrose octasulfate is removed using cross flow dialysis inter alia to form TA sucrose octasulfate gradient, and the loading of drug is accomplished. The principle is substantively identical to that used in ammonium sulfate gradient method. However, each sucrose octasulfate molecular has 8 acid groups and can form a tight complex with vinorelbine, and thus vinorelbine is well restrained. The plasma half-life of the thus-obtained vinorelbine liposome is up to 9.2 hours (Improved pharmacokinetics and efficacy of a highly stable nanoliposomal vinorelbine. The journal of Pharmacology and Experimental Therapeutics. 2009 Vol. 328 No. 1.). The serious concern in this method is that sucrose octasulfate is physiologically active and activates fibroblast growth factor in vivo (Structural basis for activation of fibroblast growth factor signaling by sucrose octasulfate. MOLECULAR AND CELLULAR BIOLOGY, October 2002, Vol. 22, No. 20), and induce a series of physiological effects. Therefore, the use of sucrose octasulfate as an excipient for injection may have a great risk.
Alza company of US uses hydrogenated soybean phosphatidyl choline (HSPC), cholesterol and DSPE-mPEG as lipid membrane, uses polyanion polymer, such as dextran sulphate, proteoglycan sulphate and cellulose sulphate, in inner water phase. Then cross flow dialysis is used to replace outer phase and form a polymer gradient, and the drug loading is accomplished. The principle is similar to that used in ammonium sulfate gradient method. This method has the aim of forming a tight complex of polyanion polymer with topotecan, and thus the drug is well restrained. The disadvantage of this method is also that the polyanion polymers are physiologically active and difficult to be metabolized in vivo, so the safety thereof shall be further investigated (Liposome-entrapped topoisomerase inhibitors. U.S. Pat. No. 6,465,008B1).
It is known from the above that the investigations of liposomes of weak alkalescent drugs, such as vinorelbine and topotecan focus on pH gradient method, general ammonium sulfate gradient method and complexation gradient method. However, they are only tested in laboratory and the materials used have safety risk: (1) the polyanionic salt, such as triethylamine salt of sucrose octasulfate and sulfate polymer, used in the above investigations are all physiologically active, and do not meet the requirement that an excipient should be inactive of physiology and of pharmacology; (2) copper ion, nickel ion, manganese ion used in the above complexation gradient method are all heavy metal ion, and their remainder in the formulation are harmful to human. Moreover, because tumor is difficult to be cured and medication is generally a long time, the in vivo accumulation of heavy metal ion will go beyond the patient tolerance.
So it is still required to develop a novel liposome and corresponding method of drug loading.